A novel non-antimicrobial treatment of bacterial vaginosis: An open label two-private centre study.

BACKGROUND: Bacterial vaginosis (BV) is the most common cause of vaginal discharge. It is caused by an imbalance in the normal vaginal microbiota. Symptoms include an offensive odour. Standard oral or vaginal antimicrobial treatments have high immediate cure rates but almost as high recurrence rates. pHyph, a vaginal pessary, contains glucono-delta-lactone (GDL) and sodium gluconate (NaG) which restore normal pH and disrupt the associated biofilm. AIM: To investigate the clinical performance of pHyph, for both treatment and recurrence prevention. Design An open-label, single arm, multi-centre first in women study. SETTING: Two private gynaecology clinics in Skåne County, Southern Sweden. METHODS: Twenty four adult women with confirmed bacterial vaginosis received the investigational product for self-administration on days 0, 2, 4, and 6 and were assessed on day 7. Clinical cure was defined as absence of three of four Amsel's criteria (pH excluded) on day 7. Safety and tolerability were also recorded. Those not cured by day 7 received a prolonged treatment protocol. Results There were three withdrawals, two before the day 7 assessment. 18/22 (82 %) were clinically cured at day 7. The pessary was well tolerated. Recurrence rates at 14 days in patients cured at day 7 after receiving standard study treatment (n = 18) were 1/18 (5.6 %) with no additional recurrences reported at 35 days. Three of four patients not cured at 7 days received continued treatment (day 7, 9, 11, and 13), but none were cured at 14 days. CONCLUSION: pHyph has the potential for both high cure rates and a reduction in recurrence.

A t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide functions as a potent antagonist of Wnt5a-dependent melanoma cell invasion.

The influential role of Wnt5a in tumor progression underscores the requirement for developing molecules that can target Wnt5a-mediated cellular responses. In the aggressive skin cancer, melanoma, elevated Wnt5a expression promotes cell motility and drives metastasis. Two approaches can be used to counteract these effects: inhibition of Wnt5a expression or direct blockade of Wnt5a signaling. We have investigated both options in the melanoma cell lines, A2058 and HTB63. Both express Frizzled-5, which has been implicated as the receptor for Wnt5a in melanoma cells. However, only the HTB63 cell line expresses and secretes Wnt5a. In these cells, the cytokine, TGFbeta1, controlled the expression of Wnt5a, but due to the unpredictable effects of TGFbeta1 signaling on melanoma cell motility, targeting Wnt5a signaling via TGFbeta1 was an unsuitable strategy to pursue. We therefore attempted to target Wnt5a signaling directly. Exogenous Wnt5a stimulation of A2058 cells increased adhesion, migration and invasion, all crucial components of tumor metastasis, and the Wnt5a-derived N-butyloxycarbonyl hexapeptide (Met-Asp-Gly-Cys-Glu-Leu; 0.766 kDa) termed Box5, abolished these responses. Box5 also inhibited the basal migration and invasion of Wnt5a-expressing HTB63 melanoma cells. Box5 antagonized the effects of Wnt5a on melanoma cell migration and invasion by directly inhibiting Wnt5a-induced protein kinase C and Ca(2+) signaling, the latter of which we directly demonstrate to be essential for cell invasion. The Box5 peptide directly inhibits Wnt5a signaling, representing an approach to anti-metastatic therapy for otherwise rapidly progressive melanoma, and for other Wnt5a-stimulated invasive cancers.

LDL and UV-oxidized LDL induce upregulation of iNOS and NO in unstimulated J774 macrophages and HUVEC.

Oxidized low-density lipoprotein (LDL) diminishes NO production from activated macrophages. The interaction between LDL and inactivated macrophages is neglected and controversial. This study examines the effect of LDL, 7-oxysterols and iron compounds on NO production in unstimulated J774 macrophages. J774 cells and human umbilical vein endothelial cells (HUVEC) were either incubated for 24 h with native LDL (LDL) or ultraviolet (UV)-oxidized LDL (UVoxLDL), in the absence or presence of an inducible nitric oxide synthase (iNOS)- or an endothelial constitutive nitric oxide synthase (eNOS)-inhibitor. J774 cells were also incubated with lipopolysaccharide (LPS), in the absence or presence of an iNOS- or an eNOS-inhibitor. Nitrite was analysed as a marker of NO production. The mRNA levels of iNOS were evaluated by reverse transcriptase polymerase chain reaction. LDL and UVoxLDL significantly increased NO production from unstimulated J774 macrophages. This increase in NO was accompanied by enhanced expression of iNOS mRNA, and was inhibited by the iNOS inhibitor. Furthermore, NO production was elevated and angiotensin-converting enzyme (ACE) activity was reduced in HUVEC following the exposure to LDL and UVoxLDL. In conclusion, LDL may serve as an important inflammatory activator of macrophages and HUVEC, inducing inducible nitric oxide production but diminishing ACE. After its oxidation, this function of LDL may be further enhanced and may contribute to the regulation and progression of atheroma formation.

The Wnt-5a-derived hexapeptide Foxy-5 inhibits breast cancer metastasis in vivo by targeting cell motility.

PURPOSE: An inherent problem in breast cancer treatment is that current therapeutic approaches fail to specifically target the dissemination of breast cancer cells from the primary tumor. Clinical findings show that the loss of Wnt-5a protein expression in the primary breast tumor predicts a faster tumor spread, and in vitro analyses reveal that it does so by inhibiting tumor cell migration. Therefore, we hypothesized that the reconstitution of Wnt-5a signaling could be a novel therapeutic strategy to inhibit breast cancer metastasis. EXPERIMENTAL DESIGN: We used in vitro techniques to show that 4T1 mouse breast cancer cells responded to the reconstitution of Wnt-5a signaling using our novel Wnt-5a mimicking hexapeptide, Foxy-5, in the same way as human breast cancer cells. Therefore, we could subsequently study its effect in vivo on the metastatic spread of cancer following the inoculation of 4T1 cells into mice. RESULTS: In vitro analyses revealed that both recombinant Wnt-5a and the Wnt-5a-derived Foxy-5 peptide impaired migration and invasion without affecting apoptosis or proliferation of 4T1 breast cancer cells. The in vivo experiments show that i.p. injections of Foxy-5 inhibited metastasis of inoculated 4T1 breast cancer cells from the mammary fat pad to the lungs and liver by 70% to 90%. CONCLUSIONS: These data provide proof of principle that the reconstitution of Wnt-5a signaling in breast cancer cells is a novel approach to impair breast tumor metastasis by targeting cell motility. In combination with existing therapies, this approach represents a potential novel therapeutic strategy for the treatment of breast cancer patients.

Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells.

Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca2+ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3beta, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1alpha, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca2+-induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells.

A formylated hexapeptide ligand mimics the ability of Wnt-5a to impair migration of human breast epithelial cells.

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated beta-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.

Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis.

Oncogenic Wnt/beta-catenin signaling occurs in a majority of colorectal cancers. In contrast, very little is known about the role of the nontransforming Wnt protein family member Wnt-5a in those tumors. In the most common of the three colon cancer stages, Dukes B or lymph node-negative, the outcome is the hardest to predict. We searched for a predictive marker in this group and observed loss of or reduced Wnt-5a expression in 50% of Dukes B tumors. Such Wnt-5a negativity was a strong predictor of adverse outcome, with a relative risk of death of 3.007 (95% confidence interval, 1.336-6.769; P = 0.008) after 5 years in Wnt-5a-negative patients. Furthermore, the median survival time after diagnosis was 109.1 months for patients with Wnt-5a-positive primary tumors but only 58 months for those with Wnt-5a-negative primary tumors. To find a possible biological explanation for these results, we studied the invasive and poorly differentiated human colon cancer cell line, SW480, which does not express Wnt-5a protein and the Wnt-5a-expressing and moderately differentiated Caco2 colon cancer cell line. We found that the addition of recombinant/purified Wnt-5a significantly reduced the migratory capacity of SW480 cells. By comparison, equivalent treatment did not significantly alter migration in the Wnt-5a-expressing Caco2 colon cancer cell line. These findings indicate that the expression of Wnt-5a in primary Dukes B colon cancer tissue constitutes a good prognostic marker for longer survival, which can be explained by the ability of Wnt-5a to impair tumor cell migration and thus reduce invasiveness and metastasis.

Glyceryl trinitrate-induced angiotensin-converting enzyme (ACE) inhibition in healthy volunteers is dependent on ACE genotype.

Evidence concerning the importance of angiotensin-converting enzyme (ACE) genotype in cardiovascular diseases is accumulating. The aim of this study was to investigate if nitric oxide (NO), generated from glyceryl trinitrate (GTN), affects human serum ACE activity in vivo, and if so, whether this effect was dependent on ACE genotype and (or) reflected in blood pressure reduction. A tablet containing 5 mg GTN was bucally administered for 5 minutes to 17 healthy volunteers. Blood pressure (BP) was recorded, and serum ACE activity, ACE genotype, and plasma cGMP was analyzed. GTN administration significantly reduced BP only in individuals with the deletion/deletion (DD) genotype. Sixty minutes after GTN administration, serum ACE activity was reduced in individuals with the insertion/insertion (II) and insertion/deletion (ID) genotypes, but not the DD genotype. Comparing the change in ACE activity over time between the genotypes resulted in the following: II vs. DD, p < 0.01; II vs. ID, p < 0.05; and ID vs. DD, p < 0.05. There was no significant difference in plasma cGMP content neither between the ACE genotypes nor before and after GTN administration. In conclusion, GTN inhibits serum ACE in vivo in individuals with the II and ID, but not the DD genotype.